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. 2017 Jun 1;31(11):1147–1161. doi: 10.1101/gad.299420.117

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© 2017 Peter et al.; Published by Cold Spring Harbor Laboratory Press

This article, published in Genes & Development, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 6. — 4EHP requires interaction with GYF1/2 proteins to repress translation. (A) A complementation assay using the R-Luc-5BoxB-A₉₅-MALAT1 reporter and λN-HA-4EHP (either wild type or the indicated mutants) was performed in control and GYF1/2-null HEK293T cells expressing GFP-MBP or GFP-GYF2 (wild type or canonical mutant). A plasmid expressing F-Luc-GFP served as the transfection control. R-Luc activity was normalized to that of the F-Luc transfection control and set to 100% in cells expressing the λN-HA peptide. Bars represent the mean values, and error bars represent standard deviations from three independent experiments. (B) Western blot analysis showing that full-length GYF1/2 levels were strongly reduced relative to control levels in the GYF1/2-null cell line. (C) Western blot analysis showing the expression of the λN-HA-4EHP and GFP-GYF2 proteins used in the assay shown in A. (D) Tethering assay using the R-Luc-5BoxB-A₉₅-MALAT1 reporter and λN-HA-4EHP (wild type or mutants) in HEK293T cells. Samples were analyzed as described in A. (+) Binding to the GYF1/2 proteins; (+/−) reduced binding to the GYF1/2 proteins; (−) no binding to the GYF1/2 proteins. (E) Western blot showing the equivalent expression of the λN-HA-4EHP proteins used in the assay shown in D. (F) Tethering assay using the R-Luc-6xMS2-A₉₅-MALAT1 reporter and MS2-HA-GYF2 (wild type or canonical mutant) in HEK293T cells. The cells were also cotransfected with GFP-MBP and F-Luc-GFP as transfection controls. R-Luc activity was normalized to that of the F-Luc transfection control and set to 100% in cells expressing MS2-HA. Samples were analyzed as described in A. (G) Western blot analysis showing the equivalent expression of the MS2-HA-GYF2 proteins. (H) Control HEK293T cells or cells depleted of GYF1/2 (KO) were transfected with the R-Luc-ARE-A₉₀-MALAT1 reporter and plasmids expressing the indicated proteins. The F-Luc-GFP reporter served as a transfection control. R-Luc activity was normalized to that of the F-Luc transfection control and set to 100% in the absence of TTP for each cell line. (I) Western blot showing the expression of the proteins in the experiment shown in H. Note that TTP is stabilized in GYF1/2-null cells expressing GYF2. However, repression did not correlate with TTP levels but with the coexpression of wild-type GYF2 and 4EHP.